Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif.

Abstract

The Rev protein of human immunodeficiency virus type 1 is a sequence-specific RNA binding protein that is essential for viral replication. Here we present evidence that Rev is a stable oligomer both in vitro and in vivo. Analysis ofRev mutants indicates that oligomerization is essential for RNA binding and hence Rev function. The oligomerization and RNA binding domains overlap over 47 amino acids. Within this region is a short arginine-rich motif found in a large class of RNA binding proteins. Substitution of multiple residues within the arginine-rich motif abolishes oligomerization, whereas several single-amino-acid substitution mutants oligomerize but do not bind RNA. Thus, Rev's arginine-rich motif participates in two distinct functions: oligomerization and RNA binding.The human immunodeficiency virus type 1 Rev protein acts posttranscriptionally to increase selectively the cytoplasmic levels of the gag-pol and env mRNAs (1-5). A specific RNA region that is required for Rev to function (Rev Response Element; RRE) has been mapped within the gag-pol and env mRNAs (3,6,7). Rev is a sequence-specific RNA binding protein that interacts directly with the RRE (8)(9)(10)(11)(12)(13)(14). This sequence-specific RNA binding activity explains the basis for Rev's selective action. Rev is a member of a class of RNA binding proteins characterized by the presence of an argininerich motif (15,16).In NaCI/20%o (vol/vol) glycerol], cell debris was removed by centrifugation, and the supernatant was loaded directly onto a 400-ml DEAE-Sepharose CL-6B column. The 200 mM salt eluate from this column was fractionated on a 2-ml heparinSepharose affinity column. Rev protein was removed by 2 M salt, dialyzed against buffer A, and stored at -80oC. As judged by staining with Coomassie blue, Rev was >98% pure and retained sequence-specific RNA binding activity (data not shown). RESULTSRev Binds to RNA as an Oligomer. Previous studies have shown that Rev binds specifically and tightly to the RRE (8)(9)(10)(11)(12)(13)(14). To address whether Rev interacts with the RRE as a monomer, or as an oligomer, we determined the sedimentation coefficient of the Rev-RRE complex. E. coli-derived Rev was incubated with a 32P-labeled RRE-containing RNA transcript, the reaction mixture was treated with RNase A and T1, and the products were fractionated on a glycerol gradient. Each gradient fraction was analyzed on a native gel to resolve the 32P-labeled Rev-RRE complex from the RNase digestion products. The results in Fig. 1 tTo whom reprint requests should be addressed. 7734The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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